Most multi-cellular organisms are organized into tissues and organs which perform specialized functions, and the "circulatory" system has evolved to transport and circulate materials between them. In higher animals, including mammals, the circulatory system is closed, in order to improve the efficiency of transport. The flow of blood fluid through this closed cardiovascular system requires that the fluid be maintained under pressure and that the pressure be regulated in some way. The regulation of systemic arterial blood pressure requires a complex interaction of numerous factors including, for example, fluid volume and vascular elasticity.
The maintenance of normal extracellular fluid volume depends primarily on the excretion of sodium (natriuresis) and water (diuresis) by the kidneys. This is determined by both the rate at which plasma is filtered at the glomerulus and the degree to which sodium is actively reabsorbed along the renal tubule. The latter process is in part regulated by the adrenal steroid hormone aldosterone. However, it has now been established that, in addition to GFR and aldosterone, there are additional "factors" which also regulate sodium reabsorption and thus the conditions present in the circulatory system.
The report by debold and coworkers in 1981 (A. J. debold et al., Life Sci. 28:89-94 (1981)) that intravenous injection of an atrial myocardial extract into rats caused a rapid and potent natriuretic response set off a detailed search over the next several years for the sequences of peptides that were responsible for this circulatory system activity. The results of this search are summarized by debold in Science 230:767-770 (1985), in which a report is presented on a series of atrial peptides and their enzymatic cleavage products that range in length from 21 to 152 amino acids. Also in the aforementioned reference is a description of the circulating form of an "atrial natriuretic factor" ("ANF") as a carboxy terminal 28 amino acid fragment of the 126 amino acid .alpha.-ANF that is found stored in the atrial granules.
Further studies on various circulating forms of ANF indicated that three additional peptides were readily found in serum. These peptides, termed atriopeptin I ("API"), atriopeptin II ("APII") and atriopeptin III ("APIII"), were found to have various activities and potencies in both in vitro and in vivo studies (see, e.g., K. F. Siegel et al., Biochem. Biophys. Res. Commun. 120:333-338 (1984); and M. L. Michener et al., Hypertension 7:469-482 (1985)). Moreover, the significantly reduced potency of API in a variety of systems, compared to APII and APIII, indicates that processing to API may be an important mechanism of hormone activation (G. Thibault, Biochem. Biophys, Res. Commun. 123:338-344 (1984); R. G. Garcia, Biochem. Biophys. Res. Commun. 126:178-184 (1985); J. G. De Mey, J. Pharmacol. Exp. Therap. 240:937-943 (1986)).
After the initial reports describing the various peptides, it became clear that inhibition of a specific cleavage enzyme could have medical applications due to its ability to increase the lifetime of circulating atrial peptides. One enzyme in particular, neutral endopeptidase 24.11 (also known as "enkephalinase") has received considerable attention in light of its presence in kidney brush border membranes, and its ability to cleave atrial peptides between the Cys-Phe linkage present inside the disulfide ring (G. M. Olins et al., Eur. J. Biochem. 170:431-434 (1987); S. R. Stephenson et al., Biochemistry 243:183-187 (1987); J. L. Sonnenberg et al., Peptides 9:173-180 (1988)). In examining the kidney as a potential source for selective degrading enzymes, with particular interest in the conversion of ANF, APII or APIII to API, the present inventors discovered a new atrial peptide-degrading enzyme isolated from bovine kidney which cleaves ANF, APII and APIII between the carboxy terminally located Ser and Phe residues. Thus, the present invention is directed in part to this newly discovered enzyme in isolated, purified form. This enzyme is referred to herein as atrial peptide-degrading enzyme, or "ADE".
The present invention is also directed to a class of novel, synthetic compounds which have been found by the inventors herein to be useful as inhibitors of ADE and are thus believed to have utility as circulatory system moderators and thus as blood pressure lowering agents. These compounds are oligopeptides or polypeptides which are characterized by a "core" of 13 or 14 amino acid residues as will be described below.